The advent of combinatorial chemistry has provided a platform for a wide variety of opportunities. The ability to produce large libraries of different compounds means that one can screen a large array of conformations, and charge distributions for their ability to bind to other compounds to act as agonists or antagonists, in binding to specific sites of a target protein, to investigate the conformation of a particular protein site, such as an enzymatic cleft or membrane channel protein, and the like.
Prior art libraries are cumbersome to screen. Difficulties arise when one attempts to identify a ligand which specifically interacts with a molecule of interest from those ligands which do not specifically interact with the molecule of interest.
Thus, there is a need to develop libraries which would provide affinity groups capable of interacting with a molecule of interest. Such libraries would be highly useful if they could simultaneously covalently bond an entity of interest to the molecule of interest to allow identification of the molecule while simultaneously releasing the affinity group for characterization and further study.
Methods and compounds are provided for directed covalent bonding of an entity to a target molecule, even site, in the presence of a plurality of chemically reactive competitive sites. Also, screening techniques are provided for identifying affinity labels for directing the entity to the target molecule. The screening technique employs a library of compounds wherein the compounds have dual functions, (1) to provide non-covalent binding affinity for a specific target site and (2) to release a tagged leaving group when a chemically reactive group on the compound covalently bonds to a reactive functionality at the target site. As such, one may combine a library of compounds with a target. By monitoring the rate at which the leaving groups are released, one can determine which leaving groups have the higher binding affinity for the target molecule. If one wishes to identify a particular region of the target molecule for binding, the members of the library which are assayed with the target compound may be individually screened, and the preferred bonding sites for the individual members determined by analysis of the product. Targets may be host derived or foreign. Host targets include cells and blood compounds present in undesirable concentrations, such as auto-, allo- or xenoreactive white cells, i.e. leukocytes, including macrophages, infected cells, platelets, tumorous cells, and overexpressed cytokines and hormones. Foreign targets include toxins, poisons, drugs of abuse, pathogenic infectious microbes, or the like.
The identified compounds can find use in enhancing the in vivo lifetime of a physiologically active entity, by selectively bonding the physiologically active entity to long lived blood component target sites in vivo. The identified compounds may also find use for altering the activity of an enzyme, including as enzyme inhibitors, for example, by providing steric hindrance at the enzyme active site, or as receptor agonists or antagonists.
The invention is further directed to combinatorial libraries wherein the library has the general structure: Biotinyl-SPhCO-A where SPhCO is a p-thiobenzoyl group and A is a moiety which includes at least one amino acid. The moiety A typically ranges from 1-20 amino acids, most typically amino acids. The moiety A can be one or more synthetic amino acid including xcex2-alanine, xcex3-aminobutyrate, O-methyl-substituted threonine, O-methyl-substituted serine, and O-methyl-substituted tyrosine.
The invention is further directed to methods of screening the combinatorial libraries of the invention. These methods typically include the steps of a) incubating a plurality of affinity molecules with a target moiety wherein each affinity molecule includes i) an affinity group including an oligopeptide and ii) a reactive functionality selected from the group including: carboxy, phosphoryl, acyl, phosphinyl, phosphonyl, imine, thioimine, ester, thioester and disulfide groups; b) binding the affinity group to the target molecule; c) forming a covalent bond between the reactive functionality and the target molecule; d) releasing the affinity group from the reactive functionality and the target molecule; and e) identifying the affinity molecule.